Immunoglobulin (Ig) gene diversification is required for an effective antibody response toward pathogens. A single protein, activation-induced deaminase (AID), triggers Ig gene diversification by the distinct processes of somatic hypermutation (SHM), class switch recombination (CSR) and, in some vertebrates, Ig gene conversion (IGC). Most evidence indicates that AID triggers these processes by deaminating Ig gene cytosines to uracils. Deamination within the expressed Ig gene variable regions can lead to SHM or IGC, and deamination within the Ig gene switch regions can lead to CSR. Differential processing of the resulting uracils is proposed to result in the distinct molecular outcomes of SHM, CSR or IGC. These observations raise several important questions: (i) how does the presence of uracils in DNA lead to the strand breaks required for IGC, CSR and possibly for SHM;(ii) how is AID specifically targeted to the Ig gene DNA (as opposed to any other cellular DNA);and (iii) does AID play a role in innate immunity by participating in the related DNA deamination-based mechanism of retroelement restriction? The first question will be answered by determining the genetic requirements for IGC and SHM in the vertebrate cell line DT40, which supports ongoing Ig gene diversification and can be readily manipulated. The answer(s) to the second question will be obtained through advanced proteomic methods to identify conserved AID-interacting proteins from DT40 and a human SHM-supporting cell line Ramos. The third question will be addressed by performing a comprehensive comparative analysis of the retroelement restriction abilities of AID proteins encoded by key branches of the vertebrate tree, from fish to humans. The fundamental studies proposed here will improve our understanding of the mechanisms of Ig gene diversification, the causes of human immunodeficiency syndromes and the origins of several B cell cancers. Relevance to public health. Antibodies are a crucial part of our immune response to pathogens such as bacteria and viruses. At the DNA level, a single protein called AID helps to mold the antibody genes in such a way that a highly effective antibody response occurs. This proposal focuses on how AID is controlled and directed within an antibody-producing cell, and thereby this research will strengthen our understanding of the antibody response and the causes of some human antibody-associated diseases including cancers.